Leishmania are obligate intracellular protozoan pathogens of humans. Within infected patients, various species of this organism inhabit and destroy macrophages within the skin or internal organs (i.e., spleen, liver and bone marrow). Thus, they cause either ulcerative, non-healing, disfiguring malignant skin lesions (e.g. L. mexicana) or degenerative and most often fatal visceral disease (e.g. L. donovani). According to World Health Organization estimates, these diseases afflict over 12 million patients annually in the Tropics and Neo-tropics worldwide. Our studies are aimed at defining the mechanisms involved in the pathophysiology of these organisms. In that regard, the basic cell, molecular and developmental biology of Leishmania and related trypanosomatid protozoa are investigated toward identifying and characterizing parasite molecules which are essential for the survival of these human pathogens. How these parasites are able to survive, access nutrients, multiply and differentiate within their insect vector and mammalian hosts are questions central to understanding the basic parasitic nature and evolutionary adaptations of these organisms. Since these parasites interact directly with their hosts, knowledge of the composition and functions of their surface membranes seems essential. To that end, unique parasite surface membrane enzymes and regulatory proteins are identified and biochemically characterized to determine their functional roles in the survival of these organisms. Further, the genes encoding such proteins are being isolated and characterized for the first time, toward defining their expression and regulation during course of parasite growth, differentiation and development. During the past year, our studies have elucidated the gene structure and chromosomal loci of several different, highly unusual Leishmania surface membrane enzymes. One of these is the unique tartrate-resistant, eternally-oriented, surface membrane acid phosphatase of Leishmania donovani (LdMAcP). In that regard, we characterized the structure of the single copy gene which encodes the LdMAcP, demonstrated its constitutive expression in both parasite life-cycle developmental forms, and determined the cell surface membrane localization of its translated product. Further, we used a variety of green fluorescent protein chimeric constructs as reporters to dissect the functional domains of this unique, tartrate-resistant parasite surface membrane enzyme. In collaboration with P.A. Bates, results of our combined biochemical and molecular studies demonstrated that another unique trypanosomatid surface membrane enzyme, a 3'-nucleotidase/nuclease and the single copy gene which encodes it have been conserved in Leishmania mexicana , an organism that produces severe cutaneous disease in humans throughout Central and South America. Our molecular analyses showed that this leishmanial 3'-nucleotidase/nuclease (3'NT/NU) is a unique member of the Class-I family of exo-nucleases. Further, our results indicated that the putative N-terminal signal peptide (SP-) functionally targeted this protein into the parasite's endoplasmic reticulum for post-translational processing and that the putative trans-membrane (TM) domain was functionally necessary for anchoring the native protein in the cell surface of parasites. In addition, our analyses demonstrated that N-linked glycosylation was not absolutely necessary for either of the enzymatic activities of the 3'NT/NU. The combined results of these studies have demonstrated both the ubiquity and structural conservation of the 3'NT/NU among all pathogenic Leishmania parasites. Various mutated-expression constructs and GFP-chimeras of these 3'NT/Nu genes were made and used to identify the specific regions of these proteins responsible for their membrane anchors, enzymatic activities and for their proper targeting to the endoplasmic reticulum and parasite cell surface. Several of these 3'NT/Nu mutated expression-constructs also proved useful for the production and purification of these normally membrane-bound parasite enzymes in a soluble form. The latter have proven useful for identifying new synthetic nucleotide inhibitors of these enzymes (collaboration with R. Johnson). Attempts to delete these 3'NT/Nu genes has uniformly resulted in their duplication elsewhere in the parasite genome indicating that they are probably essential for parasite survival. In contrast, overexpression of an anti-sense construct resulted in the total ablation of 3'NT/Nu activity in C. luciliae. Similar leishmanial anti-sense mutants will be tested for their viability both in sandflies and mice. Similar studies are in progress with the unique leishmanial surface membrane acid phosphatase. In other studies (with A. Debrabant), we demonstrated that the L. donovani calreticulin (Ld-Cal) protein functions as a lectin-like chaperone in facilitating the proper folding and processing of glycoproteins (e.g., the leishmanial secretory acid phosphatase) within the parasite's endoplasmic reticulum. Both anti-sense and overexpression constructs of the Ld-Cal are being tested to further elucidate the roles of this apparently essential protein in parasite growth and survival. Results of these studies should demonstrate whether the proteins encoded by these various genes are, in fact, essential for the survival of these pathogens within their insect-vector and/or mammalian hosts. Thus, the current studies are aimed at defining the intrinsic needs of these organisms and determining their critical nature. Results of our recent and ongoing studies continue to provide pertinent information toward understanding the unique pathophysiology of these organisms. In addition, these studies are of practical relevance toward demonstrating whether such parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs, 2) the development of new diagnostic tools and/or 3) useful as potential vaccines against these human pathogens.